Cardiovascular adaptation essential for a successful pregnancy occurs by unclear mechanisms. This proposal continues testing the hypothesis that pregnancy decreases vascular tone and contractility by enhancing sex hormone-sensitive vasodilator mechanisms. Here, we propose a new, unifying mechanism. Though estradiol and progesterone have a myriad of often nonspecific effects on components regulating blood flow, it is by altering agonist-receptor coupling to G-proteins to reduce GTPase activity that the characteristic adaptations occur. We contrast the effects of sex hormones and pregnancy on G-proteins in uterine and mesenteric arteries and the aorta to illustrate response specificity. Experiments are performed in guinea pigs over 4 years. AIM 1: Test the hypothesis that pregnancy and/or sex hormones alter G-protein activation and their mRNA expression and/or translation in a direction and magnitude consistent with the effect of pregnancy on blood flow through that artery. Photoaffinity labeling with a nonhydrolyzable, biotinylated substrate is used in subcellular fractions of arteries obtained from pregnant, nonpregnant, and estrogen or progesterone-treated castrate animals before and after NaF activation. State of the art protein chip technology will be used for some functional proteomic studies to allow micro samples. lmmunoprecipitation with specific antibody will confirm the identity of the photolabeled protein. Tissue mRNA is measured by ribonuclease protection assays and cell specific changes by RT-PCR of mRNA from pure cell samples obtained using laser capture microdissection. AIM 2: Test the hypothesis that pregnancy and/or sex hormones alters G-protein coupling to receptors of prototypic vasoactive agonists in a manner consistent with the known effect of pregnancy on blood flow through that artery. Methods described above are performed after activation of the specific receptor. AIM 3: Test the hypothesis that pregnancy and/or sex hormones decrease heterotrimeric or non-heterotrimeric GTPase activity in the arteries specifically of reproductively important organs. Photoaffinity labeling after receptor activation will be performed as described in Aim 1 except using a hydrolysable biotinylated agent. AIM 4: Test the hypothesis that pregnancy and or sex hormones decrease GTPase activity by altering GTPase activating proteins (GAPs): RGSs (regulatory G-protein subunits) for heterotrimeric G-proteins or the GAPs for small GTPases. GTP overlay and western blots are used to quantify the small GTP binding proteins and GAPs. RT-PCR of mRNA from endothelial and smooth muscle cells obtained by laser capture microdissection will localize the changes. Together, these experiments provide new understanding and avenues to elucidate pregnancy specific diseases.